Natural bone consists of collagen, HAp, and noncollagenous proteins such as proteoglycans, matrix metalloproteinases, and growth factors.11 The collagen present within bone is organized into fibers approximately 300 nm in length and 1.5 nm in diameter.11, 12 The fibers are mineralized with biological HAp, a calcium-deficient apatite with carbonate ion substitutions. The plate-like HAp crystals grow preferentially along the collagen fibers and have very small crystallite sizes with a lower crystallinity than synthetic HAp.12 They are nucleated in the 40 nm gaps present between the collagen fibers and initiated by trace proteins bound to the collagenous ECM. In addition to initiating HAp crystals, trace proteins are important for binding growth factors, remodeling the ECM, promoting angiogenesis, and inducing new bone formation.11
There are many approaches for generating hybrid constructs for bone tissue engineering that can incorporate the key aspects of the composition of bone (Table 1). One method includes coating a polymeric scaffold of micro- or nano-scaled architecture with collagen and calcium phosphate.13, 14 This type of construct is beneficial because of its simplicity and resemblance to bone ECM. The coating of both collagen and calcium phosphate may provide stem cells cultured within the construct an environment that encourages osteogenic differentiation and bone-like ECM deposition. On the other hand, such constructs may lack the complex organization and composition needed to provide cells with the biological cues to form bone. The fibrillar structure of collagen or the inclusion of growth factors may be necessary to form bone correctly. Another approach is to incorporate components of decellularized tissues within a polymeric carrier or scaffolding material.15, 16 The use of decellularized tissue may allow for the inclusion of many of the proteins and minerals found in the biological tissue. These decellularized tissues also retain much of their original structure and may provide cells with the correct template for tissue regeneration. Nevertheless, some devitalization processes may irreversibly damage the proteases and growth factors found in the native tissue, rendering them inactive. In addition, this approach requires donor tissue, although at lower amounts with respect to autologous, allogeneic, and xenogeneic bone grafts. The same disadvantages observed with these grafts are also found in hybrid constructs incorporating biological tissues. The last method covered in this review is the creation of a cell-generated ECM coating on the surfaces of the scaffold that mimics the composition of native bone.17, 18 Osteoblasts or osteogenically differentiated stem cells are typically used to generate the ECM coating. This cell-generated ECM coating may potentially have all the components that can regulate the composition and organization of the ECM similar to that of native bone. However, disadvantages to this method are that the biological components of this construct are difficult to characterize and the optimal cell culture time to generate an osteogenic ECM coating must be elucidated. In addition, it is difficult to provide the cells with the correct distribution and environment to evenly deposit the osteogenic ECM coating throughout the scaffold.
2.1. Polymeric Constructs Incorporating Collagen and Calcium Phosphate
These hybrid constructs seek to mimic bone ECM by combining porous polymeric scaffolds that have nano- and micro-sized features with collagen and calcium phosphate (Figure 1). Also under consideration are hybrid constructs incorporating gelatin instead of collagen. Although, gelatin is a hydrolyzed form of collagen, it has a similar composition, allows for cell adhesion, and is biodegradable in vivo.19–21
Figure 1. Scanning electron micrograph of a hybrid construct combining a synthetic material with collagen and nanohydroxyapatite. The construct was generated by initially electrospinning a PLLA/PBLG/collagen solution followed by 3 cycles of soaking in a calcium chloride solution then in sodium phosphate dibasic solution. The result was hydroxyapatite crystals covering collagen-like fibers. Reproduced with permission.34 Copyright 2012, Elsevier.
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Three major methods of construct synthesis have been reported in the literature. The first method involves a polymer scaffold coated initially with collagen and subsequently with hydroxyapatite. The second method uses a combination of polymer, collagen, and calcium phosphate in suspension to generate a hybrid construct after the removal of the solvent. The third method combines the two prior methods by incorporating either the collagen or the calcium phosphate into the polymer solution, removing the solvent, and then coating the surface of the composite scaffold with the other component.
In the first method, the generation of the synthetic portions of several hybrid constructs was accomplished in several ways, including electrospinning, 3D printing, and freeze-drying of polymer solutions.13, 14, 22 Electrospinning creates a non-woven fiber mesh mat with controllable fiber diameter, porosity, and thickness.23 With 3D printing, it is possible to generate a scaffold with a pre-determined macrostructure and microstructure.24 Freeze-drying of polymer solutions can create a porous sponge with a controlled pore structure.25 Each of these methods is capable of creating a highly porous scaffold that allows for the penetration of the coating solutions throughout the scaffold.
Following fabrication, the scaffold may be submerged within a collagen or gelatin solution and subsequently in simulated body fluid (SBF) solution to generate a coating of collagen or gelatin and HAp on the polymer surface. A layer-by-layer method may be used to control the thickness of the coating.26 As an example, Li et al. coated a poly(ϵ-caprolactone) (PCL) scaffold several times, first with gelatin followed by a number of layers of poly(styrene sulfonate) and finally with gelatin.13 The amount of HAp present on the scaffold may also be controlled by varying the incubation time within the SBF solution. SBF has nearly the same ionic concentration as human plasma but is highly supersaturated with respect to apatite.27 As a result, SBF forms bone-like HAp crystals on bioactive surfaces such as collagen or gelatin.27 However, SBF with the same concentration as human plasma (1X SBF) may take more than 16 days to fully coat a surface with HAp.28 Therefore, in order to decrease the mineralization time, SBF with up to 10 times the concentration of ions found in 1X SBF may be used. To further decrease the construct preparation time, it is possible to soak scaffolds in a combined collagen and SBF solution. Yun et al. used this combined method and were able to remove a fully coated construct after 24 hours.14
The second method uses electrospinning, lyophilization, or vacuum evaporation to remove the solvent from a polymer, collagen, and calcium phosphate suspension. As an example, Zhang et al. dispersed chitosan, bovine collagen, and HAp nanoparticles in dimethyl sulfoxide and acetic acid and created a nanofibrous scaffold by electrospinning.29 Lyophilization was used to generate sponges from a suspension of micro-sized HAp with chitosan and gelatin or a suspension of alginate, porcine gelatin, and β-tricalcium phosphate (TCP).30, 31 In another method, a porous sponge was generated via lyophilization using ice microparticles as a porogen.32 Specifically, Li et al. created ice microparticles from a solution of bovine collagen with dispersed 500 nm sized HAp particles frozen in liquid nitrogen. The ice microparticles were combined with poly(L-lactic acid) (PLLA) dissolved in dioxane at –5 °C, kept in liquid nitrogen for 12 hours, and lyophilized to remove the solvents. Li et al. also created porous scaffolds through vacuum evaporation. They combined HAp nanoparticles with bovine collagen and paraffin microspheres in water and malonic acid, allowed the mixture to air dry, and followed by cross-linking of the collagen using formaldehyde. Subsequently, poly(lactic-co-glycolic acid) (PLGA) dissolved in pyridine was drop-cast into the interspace between the paraffin microspheres. The pyridine solvent was allowed to evaporate under low vacuum and then the paraffin microspheres were dissolved with cyclohexane, resulting in composite scaffolds.33
In the third method, constructs are typically fabricated by electrospinning or 3D printing of a polymer and either collagen or calcium phosphate solution followed by the coating of the scaffold with calcium phosphate or collagen, respectively. A nanofibrous polymer scaffold was generated by electrospinning a combination of PLLA, poly(benzyl-L-glutamate) (PBLG), and collagen dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP).34 The nanofibrous scaffold was then coated with nano-sized crystals of HAp using 3 cycles of dipping in calcium chloride followed by dipping in sodium pyrophosphate. Another hybrid construct was generated by electrospinning PLGA combined with amorphous calcium phosphate and collagen I.35 The collagen within the scaffolds was cross-linked with glutaraldehyde and then incubated in SBF to create a HAp coating. 3D printing was used to generate a PLGA and TCP composite thumb-shaped scaffold, with multiple 1 mm by 1 mm channels present throughout.36 The scaffold was subsequently coated with a collagen-based hydrogel containing human mesenchymal stem cells (MSCs).
2.2. Biological Tissue ECM-Based Construct
Biological tissue ECM-based constructs generally consist of a polymeric carrier material and acellular biological tissue (Figure 2). Most of these hybrid constructs incorporate demineralized bone matrix (DBM) because of the osteogenic factors known to be present within DBM.16, 37, 38 However, some constructs use acellular bone matrix (ABM) or acellular urinary bladder submucosa (UBS).15, 39
Figure 2. Scanning electron micrograph of a hybrid construct composed of biological tissue ECM and a polymer, which incorporates ABM with PEG-PCL-PEG at 20 wt%. The arrows indicate a few of the ABM particles present within the construct. Reproduced.39
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DBM is an osteoinductive material that is generated by decellularizing and demineralizing cortical bone.40 A typical method of generating powdered DBM involves first cleaning of cortical bone to remove any remaining soft tissue followed by rinsing with a saline solution.40, 41 The bone is subsequently cut into small fragments and defatted and dehydrated using a 1:1 chloroform-methanol solution. The resulting fragments are frozen and pulverized into particles of sizes in the sub-millimeter range using a mill or mortar and pestle. The particles are then demineralized using hydrochloric acid ranging from 0.1–0.6 N at 4 °C and sterilized using ethylene oxide.41 ABM is generated in a similar manner as DBM, but there is no demineralization step. Instead it is generated by sterilizing the bone particulates with ethylene oxide immediately following the pulverization step.39
UBS is generated from the submucosal layer of the smooth muscle layer of the bladder. One method of creating UBS is by mechanical delamination of the submucosa from the smooth muscle followed by a treatment with dilute peracetic acid and deionized water to render the tissue acellular.15 The acellular tissue is subsequently lyophilized and pulverized using a mortar and pestle to create a powder of particulates in the range of 100 to 500 μm.15
Each of these decellularized tissues provides many of the native components of the tissue. UBS is composed mainly of collagen, but also retains fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF).42 ABM contains both the organic matrix and the mineral components of bone whereas DBM only retains the organic matrix. However, it has been shown that demineralization of bone matrix increases access to the bone morphogenic proteins (BMP) bound to the organic matrix.43 The presence of BMPs has been shown to induce bone formation at an ectopic site.44 Thus, DBM should provide any seeded cells with access to BMPs. Nevertheless, it is possible to excessively demineralize the tissue and deplete the BMPs from DBM.45 In addition, it has been shown that particulate size can affect the osteogenicity of DBM.46–48
The formed particulates from different tissues are combined with either a liquid polymeric carrier that later solidifies to form a gel or combined with a polymer to form a film. In the liquid polymeric carrier case, there are several types of polymers used and different methods of solidification employed. For example, poly(ethylene glycol)-PCL-poly(ethylene glycol) (PEG-PCL-PEG) co-polymer was dissolved in water at 60 °C, mixed with ABM, and cooled to form a composite gel.39 As another example, Kurkalli et al. combined rat DBM with Pluronic F-127, a reverse thermo-responsive polymer, and placed the solution in vivo to gel.38 Reverse thermo-responsive polymers display low viscosity at room temperature, but form a gel at body temperature.49 In another study, porcine UBS and a sucrose polymer were combined with PLGA in solution, polymerized, and the sucrose polymer was dissolved away to form a porous structure.15
In order to generate a film, a polymer and the acellular tissue particulates are combined and placed at the bottom of a well to generate a 2D surface that is a composite of the two materials. Thomas et al. combined DBM particles with PLLA beads ranging from 0.52 mm to 1.91 mm in size at varying ratios to generate a 2D substrate.16 A film was generated by combining human ABM or human DBM with PLGA in chloroform. The suspension was then cast as a thin layer in a petri dish and subsequently dried under air flow for 24 hours to create a composite thin film.37
2.3. Cell-Generated ECM-Based Construct
Cell-generated ECM-based constructs are generated by culturing stem cells, osteoblasts, or pre-osteoblastic cells on porous scaffolds. The goal of this approach is to create a cell-generated ECM coating on the surfaces of the scaffold that mimics the composition of native bone (Figure 3). The cell culture to generate the ECM has been performed under static conditions, flow conditions, electromagnetic stimulation, or dynamic strain.
Figure 3. Scanning electron micrograph of a hybrid construct which has a cell-generated ECM coating on a fiber mesh scaffold. Rat MSCs were seeded onto titanium fiber mesh scaffolds and cultured in osteogenic media for 16 days to generate the ECM visibly coating the fibers and filling the space in between. Reproduced.68
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Static culture has been used to generate ECM coatings on scaffolds because of the ease of culture. This method of culture works well for small scaffolds, where diffusional limitations of nutrients are less significant.50 The varieties of scaffolds that have been used in static culture include mineral pellets, porous polymer scaffolds, gelatin cryogels, and fiber mesh scaffolds ranging in thicknesses from 0.8 mm to 5 mm.17, 18, 51–57 Osteoblast-like cell lines such as SaOS-2 cells, bone marrow stromal cells (BMSCs) from human and rat sources, and primary rat osteoblasts have been cultured on these scaffolds from a minimum of 16 days up to 6 weeks to generate the bone mimetic ECM.
However, in large constructs, portions of the scaffold may encounter a lack in nutrients due to diffusional limitations, causing cells present in these areas to become less active.58 Bioreactor culture addresses the diffusional limitations by enhancing the mass transfer of nutrients and oxygen and the removal of metabolic waste products using fluid flow around or through the scaffold.58 In addition, fluid flow through the pores of constructs stimulates seeded cells in the form of shear stress, which has been shown to enhance osteogenic differentiation of stem and progenitor cells.59, 60
Bioreactors involving flow culture conditions include a flow perfusion bioreactor, a rotational oxygen-permeable bioreactor, and a spinner flask bioreactor. A flow perfusion bioreactor consists of a pump that perfuses constructs with media through a confined fluid path at a controlled flow rate.61, 62 A variety of porous scaffold types have been placed within a flow perfusion bioreactor including foam and fiber mesh scaffolds.63–77 The cells cultured under flow perfusion conditions are similar to those cultured under static conditions and include SaOS-2 cells and BMSCs from human, rat, and goat sources and have been cultured from 15 days up to 40 days. A rotational oxygen-permeable bioreactor consists of a rotating apparatus and a chamber which allows for gas exchange.78 Cell seeded constructs and media are placed within the chamber and rotated at a controllable rate, which causes the constructs to be continuously in free fall and thus subjected to constant fluid flow.58 Electrospun polymer fiber mesh scaffolds and polymer foam scaffolds have been cultured with rat BMSCs, rabbit amniotic MSCs, and porcine bone marrow progenitor cells using the rotational oxygen-permeable bioreactor for durations ranging from 10 days to 34 weeks.79–82 The spinner flask bioreactor generates fluid flow by suspending constructs within a media reservoir and placing a stir bar at the bottom to stir the media at a controlled rate.83 A cell-generated ECM construct was created from silk fibroin scaffolds seeded with human BMSCs and cultured within a spinner flask that was stirred for 5 weeks.84
Analogous to shear stress, pulsed electromagnetic fields (PEMF) have been shown to stimulate osteogenic differentiation of stem cells and ECM mineralization.85, 86 Additionally, dynamic loading has been shown to enhance matrix production and osteogenic differentiation.87, 88 Both PEMF and dynamic loading have been used to generate an ECM coating on constructs without the addition of any osteogenic cell culture supplements.88–94 Polymer foams, gelatin cryogels, and titanium disks were used as scaffolds and cultured with such cells as the SaOS-2 cell line and human BMSCs.89–93 These cell-seeded constructs were statically cultured for 22 days or 6 weeks in the presence of an electromagnetic field and, in some cases, with additional ultrasonic stimulation. Investigators have also cultured polymer foam scaffolds with cells such as human MSCs and an osteoblastic cell line, MLO-A5, under 5% strain for 19 or 20 days to enhance ECM production.88, 94
Also of note, several of the cell-generated ECM-based constructs were decellularized prior to analysis. Most hybrid constructs underwent 3 cycles of freezing in liquid nitrogen and thawing in 37 °C water followed by ultrasonication for 10 minutes.18, 51, 64, 73, 74, 76 An alternate method of decellularization was accomplished by treating the constructs with 0.5% Triton X-100 and 20 mM ammonium hydroxide for 3 minutes at 37 °C.57
2.4. Compositional and Physical Characterization of Hybrid Constructs
While the method of synthesis for these various hybrid constructs drastically differs, the manner of characterization is quite similar. Construct characterization has been approached using i) visualization of the distribution of cells, proteins, and minerals through the construct, ii) analysis of the protein and mineral composition, and iii) determination of physical characteristics.
Scanning electron microscopy (SEM) allows for the visualization of micro- and nano-scaled features on the surface of the construct. However, SEM does not allow for ready observation of the distribution or identification of the cellular, protein, and mineral components within the interior of hybrid constructs. A combination of fluorescent staining and confocal microscopy has been used to demonstrate the distribution of cells throughout the construct.52, 66 For further characterization of the biological factor distribution within the construct, several histological stains have been used, including methylene blue and hematoxylin & eosin for cells, alcian blue and Safranin O for proteins and glycosaminoglycans (GAGs), and alizarin red and von Kossa for minerals, while immunohistochemistry has been used to visualize the distribution of specific biological components.17, 30, 82, 84
In order to determine more precisely the composition of the hybrid constructs, the amount of proteins and GAGs has been determined using colorimetric assays and their identification has been established using enzyme-linked immunosorbent assay (ELISA), western blotting, and mass spectrometry. Typically, to determine the amount of proteins and GAGs in the construct, a detergent or chaotropic solution has been used to solubilize the proteins.17, 55, 92 Once the proteins are in solution, colorimetric assays such as the bicinchoninic acid (BCA) assay, coomassie blue assay, 1,9-dimethylmethylene blue (DMMB) assay, and chloramine T assay have been used to determine protein and GAG amounts.14, 57, 92 Precise identification of the proteins and GAGs present within the protein solution can be performed via ELISA and western blotting using antibodies.95, 96 Another method for precise identification of proteins in the solution combines a liquid chromatography column along with a tandem mass spectrometer (LC-MS/MS) and a protein database search engine.97 The protein solution first undergoes trypsinization and is then run through the liquid chromatography column, followed by injection into a tandem mass spectrometer. The resulting spectrum is analyzed by a protein database search engine and matched to specific proteins.97 If known amounts of the specific protein are analyzed in a similar manner, a standard curve can be created from the LC-MS/MS spectra and the amount of the specific protein can be calculated.98
However, each of these methods has a threshold necessary to correctly identify and determine the amount of a protein and a GAG. The chloramine T, DMMB, and BCA assays are typically able to detect molecular concentrations in the micromolar ranges, although submicromolar ranges have been reached using various modifications.99–101 The coomassie blue staining typically can only detect greater than 10 ng of protein, but has been recently improved to detect greater than 2 ng of protein.102 Meanwhile, the techniques that use antibodies allow for the detection of proteins at much lower levels. Conventional ELISA has a detection limit in the picomolar range, but using a modified technique, detection in the subfemtomolar range has been accomplished.103 Western blotting can detect greater than 100 pg of protein while LC-MS/MS permits detection of proteins on the order of subpicograms.104, 105
When determining the mineral amount and composition within the hybrid constructs, colorimetric and spectroscopic assays have been applied. Quantifying the amount of calcium phosphate mineral present within the construct has been accomplished by the colorimetric calcium and phosphate assays.51, 76 However, these assays cannot accurately determine what form of calcium phosphate is present. This issue is addressed with several techniques including X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), NMR, energy dispersive X-ray spectroscopy (EDX), or X-ray photoelectron spectroscopy (XPS) to determine the chemical composition and crystallinity of the minerals (Figure 4).13, 22, 39 Each type of mineral presents a unique spectrum or diffractogram in each of these analyses, thus allowing for identification of the specific minerals present.106
Figure 4. A SEM micrograph (A) of the surface of a cell-generated ECM-based construct and its corresponding EDX elemental mapping (B). The overlay of calcium, phosphorous, and titanium demonstrates that the calcium and phosphorous are co-localized on the titanium construct. A color version can be found in the original article.73 Reproduced.73
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Several methods have been used to determine the physical characteristics of the constructs. Contact angle measurements can be used to quantitatively demonstrate that the surface has been altered. Water placed onto the surface of a construct forms a droplet, and the hydrophobicity of the surface can be predicted based on the angle that the droplet of water makes with the surface. This is especially useful when a hydrophobic scaffold material is coated with a hydrophilic substance such as HAp or collagen. Micro-computed tomography (μCT) and fluid replacement methodologies have been used to determine the porosity of the constructs.14, 54, 65 μCT uses X-rays to visualize sections of radio-opaque materials and uses a computer to reassemble the sections into a 3-D rendering of the construct.107 Using this representation, the interconnectivity of the pores, pore size, and porosity can be calculated.107 The fluid replacement methodologies, such as mercury porosimetry, gas pycnometry, and liquid intrusion, measure the change in initial fluid volume when the pores of the constructs are filled by the various fluids, which in turn is used to calculate the construct pore size and porosity.108